The present invention relates to the manufacture of conjugates between oligo- or polysaccharides and aminated substrates. The invention also covers the products hereby obtained.
Covalent coupling of saccharides to substrates can be made mainly in two different ways, on the one hand through so-called xe2x80x9cend-point attachmentxe2x80x9d (EPA), on the other hand by so-called xe2x80x9cmultipoint attachmentxe2x80x9d (MPA). In the first case the saccharide is coupled via its terminal reducing monosaccharide unit. In the second case, which is relevant with polysaccharides, it is coupled via several monosaccharide units inside the polysaccharide. Most of the high molecular carbohydrates present in nature, which are coupled to some other high molecular compound, are immobilized via their reducing monosaccharide unit, and such natural conjugates are for example glycoproteins, glycolipides, proteoglycines and lipopolysaccharides.
Coupling via xe2x80x9cend-point attachmentxe2x80x9d means contrary to xe2x80x9cmultipoint attachmentxe2x80x9d that the molecule in an immobilized state to a high degree maintains its natural conformation and thereby maintains its capacity to specifically interact with other molecules, such as plasma proteins, growth factors, antibodies, lectins and enzymes.
For reductive coupling to aminated substrates of polysaccharides one has tried to make use of the aldehyde functions in terminal reducing monosaccharide units. However, these aldehyde functions are present as hemi-acetals, which means that the aldehyde function has extremely low reactivity resulting in unacceptably low coupling yields in most cases.
A useful method for xe2x80x9cend-point attachmentxe2x80x9d of polysaccharides to aminated surfaces is described in U.S. Pat. No. 4,613,665.
This known method is briefly constituted by partial degradation of a polysaccharide-containing D-glucosamine or D-galactosamine units, for example heparin, with sodium nitrite in an acid environment. This results in the formation of 2,5-anhydro-D-mannose units as reducing terminal units. This reducing terminal unit carries a reactive aldehyde function, which is not engaged in any hemi-acetal formation and, accordingly the aldehyde group can in high yield by reductive amination be coupled to substrates containing primary amino groups.
This known method is associated with certain drawbacks, among which the following can be mentioned.
The method requires that the polysaccharide contains D-glucosamine or D-galactosamine units with non-derivatizing and thereby primary amino groups capable of diazotization.
In the activation the molecule is degraded which for certain applications results in lost or substantially reduced biological activity.
The method also means that only half of the molecules formed in the degradation obtain a reducing end capable of coupling.
The present invention has for a main object to provide a new method whereby the disadvantages of the prior art are eliminated or at least essentially reduced.
Yet another object of the invention is to provide a method in which the substance subject to coupling to an aminated substrate will not be degraded or damaged in connection to the coupling procedure, whereby the properties of the substance are maintained substantially unchanged after the coupling to the substrate has taken place.
Yet another object of the invention is to provide a method where all molecules are made subject to coupling, whereby the yield in the coupling reaction is substantially improved compared to the prior art.
A further object of the invention is to provide a process which involves xe2x80x9cend-pointxe2x80x9d coupling to aminated substrates.
For these and other objects which will be clear from the following disclosure there is provided through the present invention a method of preparing conjugates by means of covalent coupling of a polysaccharide selected from heparin, heparin derivatives, at least partially deacetylated dermatan sulphate and dextran sulphate and a solid substance containing primary amino groups. Accordingly, the method according to the invention is a method of heterogeneous character characterized by the following steps:
a) reduction of the saccharide so that its terminal monosaccharide unit is converted to an alditol;
b) periodate oxidation of the alditol formed in step a) to the formation of a terminal aldehyde group under cleavage of the monosaccharide unit between two vicinal groups selected from hydroxyl and amino groups; and
c) reductive coupling of said alditol via the aldehyde group to the amino group of the solid substrate.
The reduction in step a) above is suitably carried out with the reducing agent selected from borohydrides, catalytic reducing agents and Raney nickel. It is particularly preferred to carry out the reduction in step a) with a borohydride, especially sodium borohydride or potassium borohydride.
With regard to the oxidation in step b) above it is suitably carried out with periodic acid or a sodium or potassium salt thereof.
The reductive coupling in step c) above is suitably carried out with a cyanoborohydride, for example sodium cyanoborohydride or potassium cyanoborohydride.
A preferred embodiment of the method according to the invention thus means that the reduction in step a) is carried out with sodium borohydride, the oxidation in step b) is carried out with sodium periodate and the reductive coupling in step c) is carried out with sodium cyanoborohydride.
The method according to the present invention is particularly suited to be used in connection with polysaccharides possessing biological activity. Thus, the polysaccharide may be selected from heparin, heparin derivatives, at least partially deacetylated dermatan sulphate and dextran sulphate.
Particularly preferred polysaccharides are heparin and heparan sulphate.
With regard to the character of the substrate there may be mentioned as examples of substrates aminated plastic objects, for example having surface coatings of polyethylenimine or chitosan. Preferred surface coating is chitosan which in its capacity as a deacetylated chitin contains primary amino groups suited for the coupling techniques involved in the present invention.
The invention also includes conjugates made by the method defined above.
The reactions involved in the method according to the present invention will now be further elucidated for a better understanding of the reaction mechanisms.
The periodate oxidation in step b) above involves the formation of a cyclic ester which is then cleaved and inter alia results in two aldehyde functions according to formula I below. 
Cleavage of vicinal dioles by periodate oxidation is previously known but as far as is known it has not previously been applied to saccharide immobilization in accordance with the present invention. In the reaction steric factors have a decisive influence on the rate by which different vicinal dioles are cleaved by periodate.
Thus, vicinal OH-groups in furanosides and pyranosides which have cis-orientation are more rapidly cleaved than such having trans-orientation. Certain trans-dioles in rigid systems are even resistant to periodate oxidation. Acyclic dioles are generally oxidized more rapidly than cyclic compounds, and such of threo-configuration are more rapidly oxidized than such with erythro-configuration.
The invention will even if it is not restricted hereto be exemplified in the following with reference to heparin. Formula II below describes a representative part of the interior of the heparin molecule. The left D-glucosamine unit wherein R is acetyl, cannot be oxidized with periodate. Of the other units only the glucuronacid unit is capable of oxidation but contains, however, dioles oriented trans-di-equatorially and difficult to oxidate. 
The major part of the molecules in native heparin having a molecular weight of between about 7000 and about 25000 contains terminally the following sequence:
- - - 4) -xcex1-D-GlcpNR- (1 - - - 4) -xcex2-D-GlcA,
wherein R is acetyl or xe2x80x94SO3H.
The terminal group can be reduced to the alditol of formula III below, the pH value suitably being maintained so high as not to lactonize the acid. By cautious periodate oxidation the threo-glycol grouping is preferably oxidized and a compound of formula IV containing a terminal aldehyde group will be obtained. 
The reaction can possibly proceed further to the formation of an aldehyde function of C-atom 5.
The relevant polysaccharides are then coupled to a solid substrate containing primary amino groups by reductive amination in accordance with formula V below. 
In an analogous manner for example chitosan can be immobilized by xe2x80x9cend-pointxe2x80x9d -coupling according to formula VI below. 
Here vicinally oriented amino and hydroxyl groups are oxidized by periodate according to formula VII below. 
The reactions involved in the present invention are by formulae shown on the appended scheme. P within a ring designates the polymer or the polysaccharide, whereas only the structure of the terminal unit has been illustrated. COOH in 6-position refers to polysaccharides having a terminal glucuronic acid unit, whereas CH2OH in 6-position relates to polysaccharides having glucosamine units in terminal position.
The coupling techniques according to the present invention constitute an essential contribution to the possibility of immobilizing polysaccharides to aminated solid substrates while maintaining the original properties of the saccharide, for example biologic activity.